Prophylactic agent for renal failure

ABSTRACT

The present invention provides an agent having a suppressive action against kidney glomerular disease and renal arteriosclerosis, a prophylactic agent for renal failure containing the above described agent, and functional foods expected to have a prophylactic effect for renal failure. The agent for preventing kidney glomerular disease and renal arteriosclerosis comprises Xaa Pro Pro as an active ingredient.

This application is a Continuation of U.S. application Ser. No.12/567,628 filed Sep. 25, 2009, which is a Continuation of ApplicationNo. PCT/JP2008/055073 filed on Mar. 19, 2008, and for which priority isclaimed under 35 U.S.C. §120. This application also claims priority ofApplication No. 2007-081638 filed in Japan on Mar. 27, 2007 under 35U.S.C. §119. The entire content of each of the above-identifiedapplications is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an active ingredient having an actionfor preventing kidney glomerular diseases and renal arteriosclerosis, aprophylactic agent for renal failure containing the active ingredient,as well as functional foods containing the active ingredient, whichfunctional food are expected to have a prophylactic effect on renalfailure.

In addition, the present invention relates to a method for preventingkidney glomerular diseases and renal arteriosclerosis. Further, thepresent invention relates to a method for preventing renal failure.

BACKGROUND OF THE INVENTION

Recently, the number of those who have a risk factor considered to causeheart diseases or renal diseases, such smoking, hypertension,hyperglycemia, and hyperlipidemia, have increased in some countries.There is an increasing trend in the number of patients suffering fromheart failure or renal failure. In addition, these pathologic states arenot limited to humans and the increasing trend in morbidity of suchdiseases are seen among animals that closely interact with humans, suchas dogs, cats, other companion animals and pet animals.

An example of an effective drug for treating heart failure is anangiotensin converting enzyme inhibitor (ACEI), such as Enalapril, whichinhibits an enzyme converting angiotensin I to angiotensin II having ahypertensive action (i.e. angiotensin converting enzyme; ACE), and has ahypotensive action. In addition, the antihypertensive drug reportedlyimproves the progression of renal damage at the same time as loweringblood pressure (J. Clin. Invest., 77, 1993-2000, 1986). Yet on the otherhand, for heart failure induced by various primary diseases, the ACEinhibitor is not effective for all cases. Rather there is a risk ofdeveloping acute renal failure accompanying lowering blood pressure. Ithas been pointed out that careful administration is required (SaishinIgaku, 48:1404 to 1409, 1993), That is, the antihypertensive drug can beinsufficient for the prophylaxis of heart failure but also could ratherinduce renal failure.

Renal failure generally refers to conditions where the kidney cannotfilter blood to remove metabolic wastes in the blood successfully.

Renal failure is the pathologic state where renal functions aredeteriorated by various renal diseases. Examples of the primary diseaseinclude diabetic nephropathy, chronic glomerulonephritis,nephrosclerosis, polycystic kidney, chronic pyelonephritis, rapidlyprogressive glomerulonephritis, SLE nephritis, glomerulosclerosis, andrenal artery blood flow failure by renal arteriosclerosis. It has beenreported that ACE inhibitors are effective against renal failure, whichhas drawn attention. Yet it has pointed out that the ACE inhibitorscould cause acute renal failure and aggravation of renal damage. Andthus it cannot be said that usefulness of these drugs is satisfactory(Saishin Igaku, 48:1404-1409, 1993). As described above, because renalfailure is caused by various factors including hyperglycemia and drugdamage, improving hypertension alone is not sufficient to prevent allrenal failures originated from the above described primary disease. Onthe other hand, glomerular disease is a common lesion of renal failureand renal arteriosclerosis is a lesion associated with renal failure.Under such circumstances, a compound having a suppressive action forglomerular disease or a compound additionally having a suppressiveaction for renal arteriosclerosis can be said to be useful forprophylaxis and/or treatment of renal failure.

Meanwhile, it has been reported that a peptide originated from foodmaterials, such as casein has the ACE inhibitory activity and suchpeptides are known to have a hypotensive action. However, whether or notthe peptides have an action for preventing kidney glomerular diseasesand an action for suppressing renal arteriosclerosis has not beendirectly demonstrated (Japanese Patent Publication No. 2782142, J. DairySci. 1995, 78:777-783, J. Dairy Sci. 1995, 78:1253-1257, Am. J. Clin.Nutr. 1996, 64:767-771). Additionally, as described above, since theprophylactic and therapeutic effect for renal failure mediated by ACEinhibition is limited, prophylaxis and treatment of the disease whichare not dependent on the ACE inhibition are desired.

SUMMARY OF THE INVENTION

The present invention provides a compound or composition having asuppressive action against kidney glomerular disease and a suppressiveaction against renal arteriosclerosis independently of the ACEinhibitory activity.

In addition, the present invention provides a prophylactic agent forrenal failure and functional foods expected to have a prophylacticeffect on renal failure containing the above described compound orcomposition.

The present invention provides a method for suppressing kidneyglomerular disease and a method for suppressing renal arteriosclerosis,which method includes administration of the above described compound orcomposition to a subject.

Accordingly, the present invention provides a method for preventingrenal failure, which method includes administration of the abovedescribed compound or composition to a subject.

The present invention is also a use of the above described compound orcomposition in producing a pharmaceutical for suppressing kidneyglomerular disease and for suppressing renal arteriosclerosis. Inparticular, the present invention is a use of the above describedcompound or composition in producing a pharmaceutical for preventingrenal failure.

The present inventors have discovered that a tripeptide having aspecific structure Xaa Pro Pro (wherein Xaa is any naturally occurringamino acid) has a suppressive action against kidney glomerular diseaseand suppressive action against renal arteriosclerosis, and have reachedto invent a pharmaceutical and functional foods useful for prophylaxisof renal failure. Concrete contents of the present invention are asfollows:

The present invention provides a suppressive agent against kidneyglomerular disease and renal arteriosclerosis containing Xaa Pro Pro asan active ingredient.

the present invention also provides a prophylactic agent for renalfailure having the above described suppressive action against kidneyglomerular disease and renal arteriosclerosis.

In addition, the present invention provides functional foods containingthe above described suppressive action against kidney glomerular diseaseand renal arteriosclerosis.

The present invention provides a method for suppressing kidneyglomerular disease and renal arteriosclerosis, which method includesadministration of Xaa Pro Pro or a composition containing Xaa Pro Pro toa subject.

In addition, the present invention provides a method for preventingrenal failure, which method includes administration of Xaa Pro Pro or acomposition containing Xaa Pro Pro to a subject.

The present invention is also a use of Xaa Pro Pro or a compositioncontaining Xaa Pro Pro in producing a pharmaceutical for suppressingkidney glomerular disease and renal arteriosclerosis. In particular, thepresent invention is also a use of Xaa Pro Pro or a compositioncontaining Xaa Pro Pro in producing a pharmaceutical for preventingrenal failure

Preferably Xaa Pro Pro is Val Pro Pro and/or Ile Pro Pro.

In other embodiment of the present invention, the active ingredient XaaPro Pro is derived from an animal milk casein hydrolysate or aconcentrate thereof.

In other further embodiment of the present invention, the activeingredient Xaa Pro Pro is originated from a fermented product obtainedby fermenting a raw material containing a milk protein with a bacteriumbelonging to the species Lactobacillus helveticus.

Preferably the bacterium belonging to the species Lactobacillushelveticus is Lactobacillus helveticus CM4 strain (FERM BP-6060).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of Test 1 examining the effect of Val Pro Pro(VPP) to suppress the thickening of small- and medium-sized renalarteries wall in rats. The test was conducted using the group of ratsadministered with L-NAME (11 rats per group), the group of ratsadministered with L-NAME and Enalapril (9 rats per group), and the groupof rats administered with L-NAME and VPP (12 rats per group). The graphrepresents mean±standard error. Comparisons were demonstrated by theMann-Whitney test. The symbol (*) in the figure indicates statisticalsignificance (p<0.05).

FIG. 2 shows the results of Test 1 examining the effect of Val Pro Pro(VPP) to suppress occurrence of the rat renal glomerular lesions.Comparisons were demonstrated by the Mann-Whitney test. The symbol (*)in the figure indicates statistical significance (p<0.05).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The agent for preventing kidney glomerular disease and renalarteriosclerosis according to the present invention comprises atripeptide with a specific structure of Xaa Pro Pro as an activeingredient. The term “agent” herein is not restricted to apharmaceutical but refers to a composition such as a pharmaceuticalcomposition or food composition, or a compound such as a food additive.The term “agent for preventing kidney glomerular disease and renalarteriosclerosis” used in the present invention refers to a compound orcomposition having an action to suppress progression of kidneyglomerular disease and renal arteriosclerosis lesions, an action toimprove kidney glomerular disease and renal arteriosclerosis lesions, ora prophylactic effect on the onset of kidney glomerular disease andrenal arteriosclerosis. In addition, the “glomerular disease” used inthe present invention refers to a disease with lesions occurred in theglomerulus of the kidney, and histologically, refers to conditions whereatrophy of the glomerulus, glomerulus deterioration, glomerulosclerosis,alterations in Bowman's capsule, or the like is observed. Concreteexamples of the name of disease include minimal change nephroticsyndrome, focal glomerular sclerosis, endocapillary proliferativeglomerulonephritis (so-called acute nephritis), IgA glomerulonephritis(mesangial proliferative glomerulonephritis), membranous nephropathy,membranoproliferative glomerulonephritis, and crescenticglomerulonephritis.

The Xaa in the active ingredient Xaa Pro Pro according to the presentinvention may be any naturally occurring amino acid. Concrete examplesinclude Val Pro Pro (valine proline proline), Ile Pro Pro (isoleucineproline proline), Ser Pro Pro (serine proline proline), and Leu Pro Pro(leucine proline proline). Preferred are Val Pro Pro and Ile Pro Prowith Val Pro Pro being more preferred. As the active ingredient, theagent may contain a combination of two or more of the tripeptides XaaPro Pro, as exemplified by a combination of Val Pro Pro and Ile Pro Pro.

The active ingredient Xaa Pro Pro may be an chemically synthesizedtripeptide or naturally occurring tripeptide.

As an chemical synthetic method of tripeptide Xaa Pro Pro, a commonmethod such as solid phase method (t-Boc method and Fmoc method) andliquid phase method can be employed. For instance, the peptide may besynthesized using an automatic peptide synthesizer such as a peptidesynthesizer (PSSM-8type) manufactured by Shimadzu Corporation. Withregard to reaction conditions for the peptide synthesis and the like,those skilled in the art can arbitrarily set appropriate reactionconditions and the like, based on their technological common knowledgedepending on the synthesis method to be selected and desired tripeptideXaa Pro Pro.

Alternatively, as the naturally occurring peptide, the tripeptide may beoriginated from an animal milk casein hydrolysate or a concentratethereof, as well as may be originated from a fermented product obtainedby fermenting a food material containing a protein with fungi orbacteria such as Aspergillus and Lactobacillus.

In cases where the animal milk casein hydrolysate or the concentratethereof, or the fermented product obtained by fermenting the rawmaterial containing the milk protein with the bacterium belonging to thespecies Lactobacillus helveticus, besides the tripeptide Xaa Pro Prowhich is the active ingredient according to the present invention, freeamino acids may be contained. Furthermore, in addition to the abovedescribed peptide and free amino acids, for example, lipids, ash,carbohydrates, dietary fibers, water and the like, all of which arenormally contained in commercially available animal milk caseins or milkproteins may be contained. Additionally, as required, a part or all ofthe appropriate components among these may be taken out.

The active ingredient Xaa Pro Pro according to the present invention maybe originated from an animal milk casein hydrolysate or a concentratethereof obtained by a method of hydrolyzing an animal milk casein with agroup of enzymes yielding Xaa Pro Pro, in particular Val Pro Pro and IlePro Pro and/or a method of fermenting animal milk with Aspergillus.

Examples of the animal milk casein include cow milk, horse milk, goatmilk, and ewe milk. In particular, cow milk casein being preferablyused.

A concentration of casein when the animal milk casein is hydrolyzed orfermented is not restricted but is preferably 1 to 19% by weight inorder to efficiently produce the animal milk casein degraded product.

An example of the above mentioned enzyme group is preferably an enzymegroup (X) including a peptidase capable of cleaving between Pro and Xaaresidues at the carboxyl terminus of the Xaa Pro Pro Xaa sequence.

The enzyme group (X) is preferably a serine type proteinase havingserine in the active center or a metal proteinase having a metal in theactive center. Examples of the metal proteinase include neutral proteaseI, neutral protease II and leucyl aminopeptidase. It is preferred thatat least one type of these metal proteinases be additionally included inthat the desired hydrolysates can be efficiently obtained in a shorttime, and even in a one-step reaction. Additionally, the peptidasecapable of cleaving the above described Pro Xaa sequence is preferablyan enzyme showing the isoelectric point in the acidic region.

An example of the above described enzyme group or enzyme group (X) is anenzyme group originated from Aspergillus such as Aspergillus oryzae.Such an enzyme group includes an enzyme group which fungus cells werecultured in an appropriate culture medium and an enzyme produced wasextracted with water. Among the enzyme groups originated fromAspergillus oryzae, an enzyme group showing the isoelectric point in theacidic region is in particular preferred.

As the enzyme group originated from Aspergillus oryzae, commerciallyavailable products can be used. Examples thereof are Sumizyme FP, LP orMP(all registered trademark, manufactured by SHINNIHON CHEMICALSCorporation), Umamizyme (registered trademark, manufactured by AmanoEnzyme Inc.), Sternzyme B 11024, PROHIDROXY AMPL(all trade names,manufactured by Higuchi Shoukai Co., Ltd.), Orientase ONS (registeredtrademark, manufactured by Hankyu Bioindustry CO., LTD), Denatyme AP(registered trademark, manufactured by Nagase ChemteX Corporation) withSumizyme FP (registered trademark, SHINNIHON CHEMICALS Corporation)being preferably used.

When these commercially available enzyme groups are used, optimumconditions are usually pre-determined. Yet, conditions such as an amountof enzyme to be used and reaction time can be appropriately altereddepending on an enzyme group to be used such that the above describedcasein hydrolysate can be obtained.

An amount of the enzyme group to be added when the above describedanimal milk casein is hydrolyzed is, for instance, such that the weightratio of enzyme group/animal milk casein in a aqueous solution in whichthe animal milk casein is dissolved is not less than 1/1000, preferably1/1000 to 1/10, especially preferably 1/100 to 1/10, further preferably1/40 to 1/10.

The reaction conditions can be appropriately selected depending on anenzyme group to be used such that an intended casein hydrolysate isobtained. A temperature is usually 25 to 60° C., preferably 45 to 55° C.And pH is 3 to 10, preferably 5 to 9, particularly preferably 5 to 8.The enzyme reaction time is usually 2 to 48 hours, preferably 7 to 15hours.

Termination of the enzyme reaction can be achieved by inactivating theenzyme. Usually the enzyme is inactivated at 60 to 110° C. to stop thereaction.

After the enzyme reaction is terminated, as required, it is preferred toremove precipitates by centrifugation removal and various filtertreatments.

Additionally, as required, a peptide having bitterness and/or smell canbe removed from the obtained hydrolysate. The removal of such abitterness component and/or smell component can be done using activatedcharcoals, hydrophobic resins or the like. For instance, the removal canbe carried out by adding the activated charcoals to the obtainedhydrolysate in 1 to 20% by weight based on an amount of casein used andby allowing to react the resulting mixture for 1 to 10 hours. Removal ofthe activated charcoals can be carried out by a known method such ascentrifugation and membrane treatment process.

The thus obtained reaction mixture containing the animal milk caseinhydrolysate or the concentrate thereof can be added as is to a liquidproduct such as beverage to be use in the functional foods. In order toimprove versatility of the animal milk casein hydrolysate, the abovedescribed reaction mixture can be, after concentrated, dried to yield apowdered form.

The content ratio of Xaa Pro Pro contained in the animal milk caseinhydrolysate or the concentrate thereof is usually not less than 1% byweight, preferably 1 to 5% by weight based on a total amount of peptidesand free amino acids in the animal milk casein hydrolysate or theconcentrate thereof. By having the content ratio of not less than 1% byweight, higher actions are expected. Additionally, either when thecontent ratio of each of Ile Pro Pro or Val Pro Pro contained in theanimal milk casein hydrolysate or the concentrate thereof is not lessthan 0.3% by weight based on a total amount of peptides and free aminoacids in the animal milk casein hydrolysate or the concentrate thereof,or when the content ration of a total of Ile Pro Pro and Val Pro Pro isnot less than 0.3% by weight, high effects are expected. Further, when0.3% by weight or more of each of Ile Pro Pro and Val Pro Pro iscontained, higher effects are expected.

The active ingredient Xaa Pro Pro according to the present invention maybe also originated from a fermented product obtained by fermenting a rawmaterial containing a milk protein with a bacterium belonging to thespecies Lactobacillus helveticus. Although the bacterium belonging tothe species Lactobacillus helveticus is preferably used individually inthe fermentation, other Lactobacillus or the like may beincluded/contained to the extent that the desired effects according tothe present invention is not adversely affected.

As the bacterium belonging to the species Lactobacillus helveticus, aproteinase-producing bacterium capable of highly producing Ile Pro Proand/or Val Pro Pro is preferred. For instance, a bacterium strainshowing a U/OD590 value measured in accordance with a method by Yamamotoet al. (Yamamoto, N. et al. J. Biochem.) (1993) 114, 740), which isbased on a method by Twining et al. (Twining, S. Anal. Biochem.) 1433410 (1984)), of not less than 400 is preferred.

An example of such a preferred bacterium strain is Lactobacillushelveticus CM4 strain (Ministry of International Trade and Industry,National Institute of Bioscience and Human-Technology, 1-1-3 Higashi,Tsukuba, Ibaraki, Japan, Zip code 305, (currently, International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology, AIST, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan, Zip code 305-8566), Accession No.: FERM BP-6060, Date ofdeposit: Aug. 15, 1997) (hereinafter referred to as CM4 strain). CM4strain has been registered under the above described accession numberunder the Budapest Treaty concerning an international approval ofmicroorganism depository on patent procedures, and already patented.

The fermented product obtained by fermenting a raw material containingmilk protein with the bacterium belonging to the species Lactobacillushelveticus can be obtained by adding a fermented milk starter containingthe bacterium strain belonging to the species Lactobacillus helveticusto a raw material containing the milk protein and by fermenting themixture on appropriately selected conditions such as fermentationtemperature.

A concentrate of the thus obtained fermented product or the like may bepowdered with freeze drying, spray drying, or the like and may be usedas a powder.

The bacterium belonging to the species Lactobacillus helveticus ispreferably used as a starter with sufficiently high activities, which ispreliminarily pre-cultured. The number of the bacteria at the beginningis preferably about 10⁵ to 10⁹ cells/ml.

The fermented product obtained by fermenting a raw material containing amilk protein with the bacterium belonging to the species Lactobacillushelveticus, for example, when used for functional foods such as Foodsfor Specified Health Uses, can be also fermented with yeast inconjunction with the above described bacterium strain belonging to thespecies Lactobacillus helveticus in order to have better flavor andbetter palatability. An example of the strain of yeast is preferably thegenus Saccharomyces such as Saccharomyces cerevisiae but not restrictedthereto. The content ratio of the yeast can be appropriately selecteddepending on a purpose.

Examples of the raw material containing the milk protein include animalmilk such as cow milk, horse milk, ewe milk, goat milk, vegetable milksuch as soybean milk, and processed milk of these milk including skimmedmilk, reconstituted milk, powdered milk, and condensed milk. Cow milk,soybean milk and processed milk of these milk is preferred and cow milkor processed milk thereof is in particular preferred.

Although the solid content concentration of the milk is not particularlyrestricted, when the skimmed milk is for instance used, the solidcontent concentration of non-fat milk is usually about 3 to 15% byweight and preferably 6 to 15% by weight for better productivity.

The above described fermentation is usually carried out by standing orstirring fermentation, for example, by a method of fermenting at atemperature of 25 to 45° C., preferably 30 to 45° C., with afermentation time of 3 to 72 hours, preferably 12 to 36 hours, whereinthe fermentation is terminated when lactic acid acidity reaches 1.5% ormore.

The content ratio of Xaa Pro Pro, preferably Ile Pro Pro and/or Val ProPro in the fermented product obtained by fermenting the raw materialcontaining the milk protein with the bacterium belonging to the speciesLactobacillus helveticus is preferably 10 mg or more, preferably 15 mgor more, in terms of 100 g of a freeze dried product of the fermentedproduct.

As for a dose or intake per day of the agent for preventing kidneyglomerular disease and renal arteriosclerosis according to the presentinvention, in the case of human, usually a dose of about 10 μg to 10 g.preferably 1 mg to 5 g, further preferably 3 mg to 1 g of the activeingredient Xaa Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro may beadministrated or taken dividedly in several times in a day.

A period of administration or intake of the agent for preventing kidneyglomerular disease and renal arteriosclerosis can be adjusted in manyways in view of age of humans or animals to be administrated or toingest and/or environments for a risk factor against kidney glomerulardisease and renal arteriosclerosis of the humans or animals. The periodcan be for example usually not less than one day, preferably 7 days to365 days.

The prophylactic agent for renal failure according to the presentinvention comprises the above described tripeptide as an activeingredient.

As for a dose or intake per day of the prophylactic agent for renalfailure according to the present invention, in the case of human,usually a dose of about 10 μg to 10 g. preferably 1 mg to 5 g, furtherpreferably 3 mg to 1 g of the active ingredient Xaa Pro Pro, preferablyVal Pro Pro and/or Ile Pro Pro may be administrated or taken dividedlyin several times in a day.

A period of administration or intake of the prophylactic agent for renalfailure can be adjusted in many ways in view of age of human or animalsto be administrated or ingest and/or environments for a risk factoragainst renal failure of the human or animals. The period can be forexample usually 1 day or more, preferably 7 days to 365 days.

A method for administrating or taking in the agent for preventing kidneyglomerular disease and renal arteriosclerosis, as well as a prophylacticagent for renal failure according to the present invention is preferablyoral administration.

A form of the agent for preventing kidney glomerular disease and renalarteriosclerosis, as well as a prophylactic agent for renal failureaccording to the present invention, when used as a pharmaceutical, canbe a form of formulation for oral administration. Examples thereofinclude tablet, pill, hard capsules, soft capsules, microcapsule,powder, pellet, and solution.

In the case of preparing as a pharmaceutical, for example,pharmaceutically acceptable carrier, adjuvant, vehicle, excipient,antiseptics, stabilizing agent, binder, pH adjusting agent, bufferagent, thickener, gelatinizer, preservatives, anti-oxidant or the likecan be, as required, used. The pharmaceutical can be produced in a unitdose form required pharmaceutical formulation administration generallyaccepted.

The foods according to the present invention comprises the agent forpreventing kidney glomerular disease and renal arteriosclerosisaccording to the present invention as an active ingredient, and can be,for example, functional foods, such as Foods for Specified Health Uses,which has efficacies such as the suppressive action against kidneyglomerular disease and renal arteriosclerosis and prophylaxis for renalfailure.

An intake to attain such efficacies, for example, in the light of thefact that the functional foods are daily, continuously or intermittentlytaken for a long period of time, in the case of human, is usually about10 μg to 10 g per day in terms of an amount of the active ingredient XaaPro Pro or an amount of Val Pro Pro and/or Ile Pro Pro, preferably 1 mgto 5 g, more preferably 3 mg to 1 g. Depending on the number of intakeper day, an intake of the foods such as the functional foods per oncecan be reduced further to a lower amount than the above describedamount.

In cases where the animal milk casein hydrolysate or the concentratecontaining the active ingredient Xaa Pro Pro is used as is, in the caseof human, the hydrolysate or the concentrate thereof is preferably takenusually in 1 mg to 100 g per day, particularly in about 100 mg to 10 g.

In cases where the freeze dried product of the fermented productcontaining the active ingredient Xaa Pro Pro is used as is, in the caseof human, usually 1 to 100 g per day in terms of a dried amount of thefreeze dried product of the fermented product, in particular, about 2 to50 g is preferably taken.

A period of the intake of the foods according to the present invention,for example the functional foods, is not particularly restricted. Theintake for a long period of time is preferred. In order to attain theabove described efficacies, for example, the period can be usually notless than one day, preferably 7 days to 365 days.

The foods according to the present invention, for example the functionalfoods, comprise the agent for preventing kidney glomerular disease andrenal arteriosclerosis containing the active ingredient Xaa Pro Pro,preferably Val Pro Pro and/or Ile Pro Pro. For instance, the animal milkcasein hydrolysate or the concentrate thereof and the fermented product,all of which were obtained in the above described manner, can be addedto a variety of foods as they are or in the form of powder or granule.If necessary, a fermented product by Lactobacillus other thanLactobacillus helveticus, other ingredients used in foods includingsugars, proteins, lipids, vitamins, minerals, or flavors, or additivessuch as various carbohydrates, lipids, vitamins, minerals, sweeteners,flavors, coloring agents, texture improving agents or a mixture thereofmay be added to improve nutritional balance, flavor, and/or the like.

The foods according to the present invention, for example the functionalfoods, can be any form of solids, gels, or liquids. Examples thereofinclude fermented dairy products such as lactobacillus beverages, avariety of processed foods and beverages, dried powders, tablets,capsules, and granule. Further, additional examples can be variousbeverage, yogurts, fluid diets, jerrys, candies, retort-packed foods,tablet candies, cookies, Castella (or Japanese sponge cake), breads,biscuits, and chocolates.

The present invention will now be described in detail by way of examplesthereof, but the scope of the present invention is by no means limitedby the examples.

EXAMPLES

[Example of Peptide Synthesis]

The active ingredient according to the present invention Ile Pro Pro andVal Pro Pro were synthesized by the following chemical synthesizingmethod (Fmoc method). The synthesis was carried out by solid phasemethod using an automatic peptide synthesizer (PSSM-8type) manufacturedby Shimadzu Corporation.

As a solid phase carrier, 50 mg of a resin (SynProPep Resin®,manufactured by Shimadzu Corporation) which is a2-chlorotrityl(2-Chlorotrityl)type polystyrene resin in which prolineprotected its amino group with a fluorenylmethyloxy carbonyl group(hereinafter referred to as Fmoc for short) is bound, was used.According to the above described amino acid sequence, Fmoc-Ile,Fmoc-Pro, and Fmoc-Val (100 μmol each) which are protected their aminogroup with the Fmoc group were reacted in the order of the peptidesequence to obtain a peptide-bound resin in accordance with aconventional method.

This peptide-bound resin was then suspended in 1 ml of a reactionmixture A (10 volume % acetic acid, 10 volume % trifluoroethanol, 80volume % dichloromethane), allowed to react at room temperature for 30to 60 minutes. Thereafter the peptides were separated from the resin andthe reaction mixture A was filtered with a glass filter. Immediatelyafter the solvent in the filtrate was removed under reduced pressure, 1ml of reaction mixture B (82.5 volume % trifluoroacetic acid, 3 volume %ethyl methyl sulfide, 5 volume % purified water, 5 volume % thioanisole,2.5 volume % ethanedithiol, 2 volume % thiophenol) was added and themixture was allowed to react at room temperature for six hours touncouple the side chain protecting group. To the resultant 10 ml ofanhydrous ether was added to precipitate the peptide, and centrifuged at3000 revolutions for five minutes to separate. The precipitate waswashed with anhydrous ether was several times and then dried by sprayingnitrogen gas. The entire volume of the thus obtained unpurifiedsynthetic peptide was dissolved in 2 ml of 0.1N hydrochloric acidaqueous solution and then HPLC using a C18 reverse phase column wascarried out in accordance with the conditions below.

Pump: Type L6200 intelligent pump (Hitachi, Ltd); detector: Type L4000UVdetector (Hitachi, Ltd) for detection of ultra-violet absorption at 215nm; column: Micro Bondashere5μC18 (Waters Corp.); eluting solutions:solution A; 0.1% by weight TFA aqueous solution, solution B;acetonitrile with 0.1% by weight TFA aqueous solution; (B/A+B)×100(%):0→40% (60 minutes); and flow rate: 1 ml/minute. The eluted fractionshowing the maximum absorbance was collected and then freeze-dried toobtain the intended synthetic peptide Ile Pro Pro and Val Pro Pro (5.7mg and 6.5 mg, respectively). The purified peptide was analyzed using anautomatic protein primary structure analyzer (Type PPSQ-10, manufacturedby Shimadzu Corporation) from the N-terminal of the peptide. Thepurified peptide was further analyzed an amino acid analyzer (Type 800series, manufactured by JASCO corporation) to confirm that the peptidewas the one as designed.

[Example of Preparation of Animal Milk Casein Hydrolysates]

To Casein originated from cow milk (Nippon NZMP Ltd.) (1 g) 99 g ofdistilled water adjusted to about 80° C. was added and the mixture wasstirred well. Subsequently, IN sodium hydroxide (manufactured by WakoPure Chemical Industries, Ltd.) solution was added. The mixture was thenadjusted to pH 7.0 and a temperature of 20° C. to prepare a substratesolution.

To the obtained substrate solution a commercially available enzyme(registered trademark “Sumizyme FP”, manufactured by SHINNIHON CHEMICALSCorporation) was added, which was derived from Aspergillus oryzae andcontains at least metal protease, serine protease, neutral protease I,neutral protease II and leucyl aminopeptidase such that theenzyme/casein weight ratio of 1/25 was attained. The mixture was allowedto react at 50° C. for 14 hours. Subsequently, the enzymes wereinactivated by autoclave at 110° C. for ten minutes, thereby obtaining acasein enzymolytic product solution. Then, the obtained enzymolyticproduct solution was dried by spray drying to prepare a powder.

Components contained in the obtained powder were analyzed. Proteins weredetermined by Kjeldahi method and amino acids were measured by an aminoacid analyzer. In addition, a difference after subtracting an amount ofthe amino acids from an amount of the proteins was defined as an amountof peptides. Further, lipids, ash content and water were determined byacid decomposition method, direct ashing method, and drying method byheating under atmospheric pressure, respectively. The remainder aftersubtracting the amount of each component from 100% was defined as anamount of carbohydrates. The results showed that the powder contained35.8% by weight amino acids, 45.7% by weight peptides, 6.6% by weightwater contents, 0.2% by weight lipids, 4.1% by weight ash contents and7.6% by weight carbohydrates.

<Measurement of Amino Acids Composing the Peptide>

The powder prepared above was dissolved in an appropriate amount ofdistilled water and analyzed with an automatic peptide analyzer (tradename PPSQ-10 manufactured by Shimadzu Corporation) to check in whatorder amino acid is located from the N-terminal in the powder. Theautomatic peptide analyzer does not detect any free amino acids.

The total amount of the amino acid located at the 5th residue positionwas 120 pmol and the total amount of the amino acid located at the 6thresidue position was 100 pmol. Based on these results, most peptides inthe above described powder were found to be dipeptides or tripeptides.Additionally, a proportion of the peptides having Pro as the amino acidat the 2nd residue position markedly increased to 49.5%. A proportion ofthe peptides having Pro as the amino acid at the 3rd residue positionwas as high as 29.8%.

Hence the above described powder contains many tripeptides of Xaa ProPro. Thus these peptides were presumably a peptide highly resistant toenzymatic degradation actions of proteases in living bodies.

<Measurement of Peptides Contained in Enzymolytic Products>

For the above described powder of the enzymolytic product, amounts ofthe tripeptides shown in Table 1 contained in the powder was determined,in accordance with a conventional method, using various chemicallysynthesized standard peptides. Results are shown in Table 1.

TABLE 1 Amount of tripeptide (μg/ml) Peptide sequence in 10 mg/ml ofpowder Ser Pro Pro 2.9 Val Pro Pro 29.5 Ile Pro Pro 28.1 Phe Pro Pro27.2 Other Xaa Pro Pro 28.8

The amount of peptides and free amino acids in a solution in which theabove described powder was dissolved and diluted in distilled water was8.15 mg/ml, and an amount of the peptides was 4.57 mg/ml, an amount ofXaa Pro in the peptides was 514.5 μg. The proportion of Xaa Pro based ona total amount of the peptides and free amino acids in the powder wastherefore 6.3% by weight. Further, an amount of Xaa Pro Pro in thepeptide was 116.5 μg and it was thus confirmed a proportion of Xaa ProPro based on a total amount of the peptides and free amino acids in thepowder was 1.4% by weight.

[Example of Preparation of CM4 Fermented Milk Animal Feeds]

Using fermented milk obtained by fermenting a raw material containing amilk protein with the CM4 strain, animal feeds containing the activeingredient Xaa Pro Pro according to the present invention was prepared.

Commercially available powdered non-fat milk was dissolved in distilledwater to the solid content of 9% (w/w) and then autoclaved at 105° C.for ten minutes to sterilize under heating at high temperature. Aftercooled to room temperature, the resulting solution was inoculated with aCM4 strain starter fermentation solution (the number of the bacteria:5×10⁸/ml) at 3% (v/w) and left to stand at 37° C. for 24 hours to yieldCM4 fermented milk.

The obtained CM4 fermented milk was sterilized at a reaching temperatureof 80° C. and then freeze-dried to yield the powder. The obtained freezedried powder was mixed with commercially available powdered feeds (tradename “CE-2”, manufactured by CLEA Japan, Inc.) at the mass ratio of10:90. The mixture was formed into solid feeds to obtain CM4 fermentedmilk feeds. The feeds contained 34.1 mg/kg of Val Pro Pro and 17.1 mg/kgof Ile Pro Pro, both of which peptides were originated from the CM4fermented milk.

[Test1: Effect of Val Pro Pro (VPP) to Suppress Kidney GlomerularDisease and Renal Arteriosclerosis]

In order to evaluate the effect of the tripeptide Val Pro Pro (VPP) tosuppress kidney glomerular disease and renal arteriosclerosis, a test onthe frequency of occurrence of renal glomerular lesions and the degreeof thickening of the small and middle renal artrey wall was carried out.

The test was carried out using three groups of male Wistar rats of sevenweeks old (Japan SLC, Inc.), each of which group consist of 9 to 12rats. After acclimation for a week, the animals received: water withL-NAME (manufactured by Sigma) being dissolved at a concentration of 1g/L; water with L-NAME and VPP being dissolved at a concentration of 1g/L and 0.3 g/L respectively; or water with L-NAME and angiotensinconverting enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/Land 0.5 mg/L respectively, ad libitum for eight weeks. The dose ofEnalapril was determined such that its activity to inhibit ACE wasequivalent to VPP's.

The rats were sacrificed by exsanguination under diethyletheranesthesia. The kidney was enucleated and then fixed with 10% neutralbuffered formalin solution. Tissue samples were prepared from the fixedkidney by excising in parallel with the minor axis such that the tip ofrenal papilla was included. The specimen were embedded in paraffin andcut into thin sections with a thickness of 2.0 to 2.5 μm using amicrotome. Two to five sections per rat were prepared. The thin slicedsections were subjected to hematoxylin eosin staining or periodic acidmethenamine silver staining (PAM staining). In accordance with a methodby Foglieni et al. (Chiara Foglieni, et al., “Protective effect of EDTApreadministration on renal ischemia” BMC Nephrology 2006, 7:5, [online],Mar. 15, 2006, BioMed Central Ltd,http://www.biomedcentral.com/bmcnephrol/), a histopathologicalexamination was, with keeping the name of each sample concealed, carriedout by a pathologist who was not involved in the test.

Under a microscopic observation, the glomerulus that atrophic lesions,deterioration of the glomerulus, glomerulosclerosis, alteration ofBowman's capsule or the like was observed was defined as the glomeruluswith lesions, in accordance with the definition of the glomerularlesions described in a book by Enomoto et al. (edited by Makoto Enomoto,Yuzo Hayashi, and Hisako Tanaka, “Pathological tissues of experimentalanimals”, Softscience, pages 380-396). The atrophic lesion refers toconditions where the size ratio between the glomerulus and Bowman'scapsule is altered due to glomerular atrophy. The deterioration of theglomerulus refers to conditions where the blood capillary cavity of theglomerulus is disappeared and glomerular functions are lost. Theglomerulosclerosis refers to conditions where increased mesangial matrixwas observed without accompanying hyperplasia of collagen fiber. Thealteration of Bowman's capsule refers to conditions where hypertrophy ofepithelial cells and uptake of lipid droplets and/or protein particlesare observed. In one whole section, the frequency of occurrence of theseglomerular lesions was scored from 0 to 3, and a score was determinedfor each individual rat. The thus determined score for each individualrat was subjected to statistical analysis and a score for each group wasdetermined.

Score 0: A case where one or two glomerular lesions owing to physiologicalterations were observed.

Score 1: A case where one or two focal regions in the glomerulus wereobserved, which regions were a limited and small range.

Score 2: A case where two to four focal regions in the glomerulus wereobserved, which regions were a middle sized range.

Score 3: A case where five or more focal regions in the glomerulus wereobserved in a wide range.

The focal regions herein indicate that a plural of lesions are, on thebasis of pathological findings, observed in a certain area in one wholesection.

The thickening of the small-, and mid -sized artery wall existing in therenal cortex was also evaluated by a tissue pathological test in thesame manner as the observation of the glomerular lesions. Under amicroscopic observation, all arteries observed in one whole section werecompared with arteries observed at the virtually same location in thespecimen from a normal rat of the same age in weeks. The thickness ofthe small- and mid-sized artery wall was scored from 0 to 3 as describedbelow and a score for each individual rat was determined. The determinedscore for each individual rat was subjected to statistical analysis anda score for each group was determined.

Score 0: A case where only the thickening equal to the normal rat in onewhole section was observed.

Score 1: A case where the thickening was observed in a mild degree or atlow frequency, compared with the normal rat.

Score 2: A case where the thickening was observed in a moderate degreeor at moderate frequency, compared with the normal rat.

Score 3: A case where the thickening was observed a high degree or athigh frequency, compared with the normal rat.

The obtained results are shown in FIG. 1 and FIG. 2. As shown in FIG. 1,compared with the group of rats fed with L-NAME alone, the group of ratsfed with VPP showed a decrease in the degree of thickening of the small-and mid-sized artery wall, which indicates that VPP has the effect tosuppress renal arteriosclerosis. Further, as shown in FIG. 2, the intakeof VPP resulted in suppression of the frequency of occurrence of theglomerular lesions, indicating the effect of VPP to suppress kidneyglomerular diseases. Accordingly, since VPP suppresses the thickening ofthe small- and mid-sized artery wall as well as occurrence of theglomerular lesions VPP is proven to be effective on the prophylaxis ofrenal failure. Judging from the fact that Enalapril did not show thesuppressive effect against either renal arteriosclerosis or glomerulardiseases, it was confirmed that the effect of VPP was not dependent onthe ACE inhibition

[Test 2: Effect of Ile Pro Pro (IPP) to Suppress Kidney GlomerularDisease and Renal Arteriosclerosis]

The tripeptide Ile Pro Pro (IPP) was also tested for renal pathologicstate, in order to evaluate its effect to suppress kidney glomerulardisease and renal arteriosclerosis.

The test was carried out using three groups of male Wistar rats of sevenweeks old (Japan SLC, Inc.), each of which group consist of 9 to 12rats. After habituation breeding for a week, drinking water with L-NAME(manufactured by Sigma) being dissolved at a concentration of 1 g/L;drinking water with L-NAME and IPP being dissolved at a concentration of1 g/L and 0.3 g/L respectively; drinking water with L-NAME andangiotensin converting enzyme (ACE) inhibitor, Enalapril at aconcentration of 1 g/L and 0.5 mg/L respectively, were supplied adlibitum for eight weeks. The dose of Enalapril was determined such thatits activity to inhibit ACE was equivalent to the activity of IPP toinhibit ACE.

The rats were sacrificed by exsanguination under diethyletheranesthesia. The kidney was enucleated and then fixed with 10% neutralbuffered formalin solution. Tissue samples were prepared from the fixedkidney by excising in parallel with the minor axis such that the tip ofrenal papilla was included. The specimen were embedded in paraffin andcut into thin sections with a thickness of 2.0 to 2.5 μm using amicrotome. Two to five sections per rat were prepared. The thin slicedsections were subjected to hematoxylin eosin staining or periodic acidmethenamine silver staining (PAM staining). In accordance with a methodby Foglieni et al. (Chiara Foglieni, et al., “Protective effect of EDTApreadministration on renal ischemia” BMC Nephrology 2006, 7:5, [online],Mar. 15, 2006, BioMed Central Ltd,http://www.biomedcentral.com/bmcnephrol/), a histopathologicalexamination was, with keeping the name of each sample concealed, carriedout by a pathologist who was not involved in the test.

Under a microscopic observation, the glomerulus that atrophic lesions,deterioration of the glomerulus, glomerulosclerosis, alteration ofBowman's capsule or the like was observed was defined as the glomeruluswith lesions, in accordance with the definition of the glomerularlesions described in a book by Enomoto et al. (edited by Makoto Enomoto,Yuzo Hayashi, and Hisako Tanaka, “Pathological tissues of experimentalanimals”, Softscience, pages 380-396). The atrophic lesion refers toconditions where the size ratio between the glomerulus and Bowman'scapsule is altered due to glomerular atrophy. The deterioration of theglomerulus refers to conditions where the blood capillary cavity of theglomerulus is disappeared and glomerular functions are lost. Theglomerulosclerosis refers to conditions where increased mesangial matrixwas observed without accompanying hyperplasia of collagen fiber. Thealteration of Bowman's capsule refers to conditions where hypertrophy ofepithelial cells and uptake of lipid droplets and/or protein particlesare observed. In one whole section, the frequency of occurrence of theseglomerular lesions was scored from 0 to 3, and a score was determinedfor each individual rat.

Score 0: A case where one or two glomerular lesions owing to physiologicalterations were observed.

Score 1: A case where one or two focal regions in the glomerulus wereobserved, which regions were a limited and small range.

Score 2: A case where two to four focal regions in the glomerulus wereobserved, which regions were a middle sized range.

Score 3: A case where not less than five focal regions in the glomeruluswere observed in a wide range.

The focal regions herein indicate that a plural of lesions are, on thebasis of pathological findings, observed in a certain area in one wholesection.

The thickening of the renal artery wall at the hilum existing in therenal medulla and the thickening of the small and middle artery wallexisting in the renal cortex were also evaluated by a tissuepathological test in the same manner as the observation of theglomerular lesions. Under a microscopic observation, all arteriesobserved in one whole section were compared with arteries observed atthe virtually same location in the specimen from a normal rat of thesame age in weeks. The thickening of the renal artery wall at the hilumand the thickening of the small- and mid-sized artery wall were scoredfrom 0 to 3 as described below and a score for each individual rat wasdetermined. The determined score for each individual rat was subjectedto statistical analysis and a score for each group was determined.

Score 0: A case where only the thickening equal to the normal rat in onewhole section was observed.

Score 1: A case where the thickening was observed in a mild degree or atlow frequency, compared with the normal rat.

Score 2: A case where the thickening was observed in a moderate degreeor at moderate frequency, compared with the normal rat.

Score 3: A case where the thickening was observed a high degree or athigh frequency, compared with the normal rat.

These lesions of the glomerulus, the thickening of the renal artery wallat the hilum and the thickening of the small- and mid-sized artrey wallwere individually scored. A rat with the score of not less than 1 in allthree items was defined as one having the pathologic state. Using thenumber of the rats with and without the pathologic state, comparisons bylikelihood-ratio test were made.

TABLE 2 Evaluation results on rat renal pathologic state Administratedgroup With pathologic state Without pathologic state L-NAME 4 rats 7rats L-NAME and 0 rats 9 rats IPP (*) L-NAME and 2 rats 7 rats Enalapril(*) p < 0.05

These results revealed that IPP also suppressed the onset of thepathologic state of the glomerulus and the renal artery, and thus IPP iseffective on the prophylaxis of renal failure. In addition, like in Test1, Enalapril herein did not exhibit such an effect. It was henceconfirmed that the effect was not dependent on the ACE inhibition.

Since the agent for preventing kidney glomerular diseases and renalarteriosclerosis according to the present invention shows effectiveness,even when angiotensin converting enzyme (ACE) inhibitors do not exhibitthe suppressive action, the agent is of great use as a pharmaceuticalfor the prophylaxis and/or treatment of renal failure, in particular asa prophylactic agent, which agent is not dependent on the ACE inhibitoryactivity. In addition, the active ingredient is a naturally occurringtripeptide originated from foods and the like, it is expected that thepharmaceutical with high efficacy and with little concern for sideeffects is provided.

Furthermore, by using or adding the agent for preventing kidneyglomerular diseases and renal arteriosclerosis according to the presentinvention in foods or beverages, the functional foods expected to havethe prophylactic effect for renal failure are provided.

REFERENCES

-   1. Japanese Patent No 2782142-   2. J. Clin. Invest. 77, 1993-2000, 1986-   3. Saishin Igaku, 48:1404 1409, 1993-   4. J. Dairy Sci. 1995, 78:777-783-   5. J. Dairy Sci. 1995, 78:1253-1257-   6. Am. J. Clin. Nutr. 1996, 64:767-771

1. A method for suppressing kidney glomerular disease and renalarteriosclerosis, comprising administrating Xaa Pro Pro to a subject. 2.The method for suppressing kidney glomerular disease and renalarteriosclerosis according to claim 1, wherein said Xaa Pro Pro is ValPro Pro and/or Ile Pro Pro.
 3. The method for suppressing kidneyglomerular disease and renal arteriosclerosis according to claim 1,wherein said Xaa Pro Pro is originated from an animal milk caseinhydrolysate or a concentrate thereof.
 4. The method for suppressingkidney glomerular disease and renal arteriosclerosis according to claim3, wherein said animal milk casein hydrolysate is a fermented productobtained by fermenting an animal milk casein with Aspergillus.
 5. Themethod for suppressing kidney glomerular disease and renalarteriosclerosis according to claim 3, wherein said animal milk caseinhydrolysate is a decomposed product obtained by hydrolyzing an animalmilk casein with an enzyme derived from Aspergillus.
 6. The method forsuppressing kidney glomerular disease and renal arteriosclerosisaccording to claim 5, wherein said enzyme derived from said Aspergillusis an enzyme derived from Aspergillus oryzae.
 7. The method forsuppressing kidney glomerular disease and renal arteriosclerosisaccording to claim 1, wherein said Xaa Pro Pro is a fermented productobtained by fermenting a raw material containing a milk protein with abacterium belonging to the species Lactobacillus helveticus.
 8. Themethod for suppressing kidney glomerular disease and renalarteriosclerosis according to claim 7, wherein said bacterium belongingto the species Lactobacillus helveticus is Lactobacillus helveticus CM4strain (FERM BP-6060).
 9. A method for suppressing renal failure,comprising administrating Xaa Pro Pro to a subject.
 10. The method forsuppressing renal failure according to claim 9, wherein said Xaa Pro Prois Val Pro Pro and/or Ile Pro Pro.
 11. The method for suppressing renalfailure according to claim 9, wherein said Xaa Pro Pro is originatedfrom an animal milk casein hydrolysate or a concentrate thereof.
 12. Themethod for suppressing renal failure according to claim 11, wherein saidanimal milk casein hydrolysate is a fermented product obtained byfermenting an animal milk casein with Aspergillus.
 13. The method forsuppressing renal failure according to claim 11, wherein said animalmilk casein hydrolysate is a decomposed product obtained by hydrolyzingan animal milk casein with an enzyme derived from Aspergillus.
 14. Themethod for suppressing renal failure according to claim 13, wherein saidenzyme derived from said Aspergillus is an enzyme derived fromAspergillus oryzae.
 15. The method for suppressing renal failureaccording to claim 9, wherein said Xaa Pro Pro is a fermented productobtained by fermenting a raw material containing a milk protein with abacterium belonging to the species Lactobacillus helveticus.
 16. Themethod for suppressing renal failure according to claim 15, wherein saidbacterium belonging to the species Lactobacillus helveticus isLactobacillus helveticus CM4 strain (FERM BP-6060).